Qing et al. Cell Commun Signal (2021) 19:83 https://doi.org/10.1186/s12964-021-00764-5

RESEARCH Open Access Pharmacologic targeting of the P‑TEFb complex as a therapeutic strategy for chronic myeloid leukemia Yingjie Qing1†, Xiangyuan Wang1†, Hongzheng Wang1, Po Hu1, Hui Li1, Xiaoxuan Yu2, Mengyuan Zhu1, Zhanyu Wang1, Yu Zhu3, Jingyan Xu4, Qinglong Guo1* and Hui Hui1*

Abstract Background: The positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with diferentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells. Materials and methods: mRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell diferentiation and apoptosis. Cell transfection, immunofuorescence analysis and co-immu- noprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo. Results: We reported that the anti-CML efects in K562, KU-812 and primary CML cells induced by wogonin were reg- ulated by P-TEFb complex. We also confrmed the relationship between CDK9 and erythroid diferentiation via knock- down the expression of CDK9. For further study the mechanism of erythroid diferentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apop- tosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor efects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human ­CD45+ cells in spleens of mice. We also verifed that wogonin exhibited anti-CML efects through modulating P-TEFb activity in vivo. Conclusions: Our study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment. Keywords: Wogonin, Cell diferentiation, P-TEFb, CDK9, CML

Background CML is a malignant clonal disease that originates in hematopoietic stem cells [1]. Clinically, it is divided *Correspondence: [email protected]; [email protected] †Yingjie Qing and Xiangyuan Wang have contributed equally. into three phases including chronic phase, acceler- 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory ated phase and blast phase [2]. CML patients com- of Carcinogenesis and Intervention, China Pharmaceutical University, 24 monly arise t(9;22)(q34;11) chromosomal translocation Tongjiaxiang, Nanjing 210009, People’s Republic of China Full list of author information is available at the end of the article and formation of fusion gene BCR-ABL1, which is

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transcribed and translated into BCR-ABL1 protein with targeting CDK9 is an efective tumor therapy strategy tyrosine kinase activity, thereby activating downstream [18]. signaling pathways such as PI3K-Akt-mTOR and Diferentiation induction therapy is a treatment MAPK, leading to abnormal cell proliferation [1, 3, 4]. method that uses diferentiation inducers to induce Tyrosine kinase inhibitors (TKIs) are currently the pre- malignant tumor cells to transform into mature cells. ferred drug in clinical CML patients and Imatinib is the Normal cells are less afected by diferentiation induc- frst generation of TKIs used for the treatment of CML ers due to their high degree of diferentiation. Terefore, [5]. However, although TKIs therapy can alleviate the compared with chemotherapeutic drugs with poor selec- condition of most CML patients, long-term use cannot tivity and high cytotoxicity, diferentiation inducers have eliminate the existence of factors such as leukemia stem lower side efects [19]. Diferentiation induction therapy cells and high price [6, 7]. Terefore, new ideas should has achieved good efects in the treatment of acute pro- be expanded in the development of CML drugs to solve myelocytic leukemia (APL), which is a model of cell dif- the increasing urgent problems of clinical medication ferentiation therapy [20]. However, only retinoic acid of CML. (RA) combined with arsenic trioxide has been successful Cyclin-dependent kinases (CDKs) are serine/threonine in the treatment of APL. Tus, new diferentiation induc- kinases found in yeast, which have the functions of regu- ers are needed to develop in the treatment of CML. lating cell cycle and cell transcription [8, 9]. Cycle CDKs During the process of hematopoiesis, hematopoi- (CDK1, 2, 4, 6) mainly play a role in the regulation of cell etic stem cells (HSC) frst diferentiate into common cycle, while transcriptional CDKs (CDK7-9, 10–13) act myeloid progenitor (CMP), and then CMP diferentiate on the transcriptional regulation process of cells [10]. into megakaryocyte erythrocyte progenitor (MEP) and Transcription CDKs make RNA Polymerase II (RNA granulocyte macrophage progenitor (GRA) cells. How- Pol II) carboxyl terminal structure domain (C-Terminal ever, MEP can further diferentiate into primordial mega- Domain, CTD) phosphorylation, thus promote the ini- karyocytes and erythrocytes, and then they enter the tiation and extension of transcription [11]. Inhibition of diferentiation pathways of megakaryocytes and eryth- CDKs mainly afects the accumulation of transcripts with rocytes respectively [21]. Te diferentiation of MEP into short half-life, such as anti-apoptotic family members’ megakaryocytes and erythrocytes is afected by several genes BCL2, MCL1 and XIAP, cell cycle regulating family key transcription factors, such as GATA binding protein genes CCND1 and CMYC, and tumor suppressor genes 1 (GATA-1), friend of GATA-1 (FOG-1), Runt-related TP53 [10]. Terefore, transcriptional CDKs is one of the transcription factor 1 (RUNX1), etc. [22]. GATA-1 is a potential targets of tumor therapy. transcription factor that can initiate erythroid / megakar- RNA Pol II is a transcription enzyme, which regulates yocyte diferentiation. When MEP cells are at the transi- the messenger RNA and non-coding RNA [12]. RNA tion point of erythroid / megakaryocyte diferentiation, pol II activity in the transcription cycle stages is strictly GATA-1 is activated [23]. Te selection of GATA-1 for regulated, which makes phosphorylation RNA pol II the direction of cell diferentiation is related to the active CTD in dynamic changes and drives transcription from complex formed by GATA-1 and other regulatory pro- pre-initiation, initiation, extension to termination [13]. teins. GATA-1 interacts with the N-terminal zinc fnger RNA transcription is catalyzed by CDK7 and CDK9 in structure of FOG-1 to activate the expression of eryth- turn. Firstly, CDK7 phosphorylates the serine (ser5) site roid related gene γ-globin and promote erythroid difer- of CTD of RNA pol II, thus activating RNA pol II and entiation of cells [24]. On the contrary, GATA-1 forms acting as a part of pre-transcriptional initiation complex a complex with RUNX1, which acts on the promoter to promote the initiation of transcription [14]. Subse- GPIbα of megakaryocytes and promotes the diferentia- quently, the serine (ser2) at the second position of CTD tion of megakaryocytes, and the process of diferentiation of RNA pol II is phosphorylated by CDK9, which pro- highly dependents on P-TEFb [25]. Tus, P-TEFb plays motes the transcription process into the extension stage an important role in megakaryocyte/erythroid transi- [15, 16]. P-TEFb is a kinase complex formed by catalytic tion point and is the key regulatory molecule of direc- subunit CDK9 and regulatory subunit cyclin T1. Under tional diferentiation. Inhibition of CDK9 kinase activity the action of catalytic subunit CDK9, ser2 of CTD of can inhibit the activity of P-TEFb, weaken the interaction RNA pol II is phosphorylated, which promotes RNA between GATA-1 and RUNX1 protein, and inhibit mega- transcription extension [17]. Terefore, P-TEFb plays karyocyte diferentiation, thus promoting erythroid dif- an important role in the production of cellular mRNA. ferentiation of cells. CDK9 inhibitors inhibit the transcription of downstream Wogonin (5,7-dihydroxy-8-methoxy-2-phenyl- genes by reducing the phosphorylation of RNA pol II 4h-1-benzopyran-4-one) is one of the bioactive com- by inhibiting the formation of P-TEFb [15]. Terefore, ponents isolated from the Chinese herbal medicine Qing et al. Cell Commun Signal (2021) 19:83 Page 3 of 16

Scutellaria baicalensis, which has anti-oxidant, anti- protocols. Te fuorescence was detected by fow cytom- cancer, anti-infammatory and other pharmacological etry (Becton–Dickinson Biosciences, New Jersey, USA). activities [26, 27]. Here, we investigated P-TEFb kinase and the data analysis was performed by Flowjo software. activity in regulating diferentiation in CML cells. Moreo- ver, we verifed that anti-proliferation activity of wogonin Flow cytometric analysis of cell diferentiation involved in P-TEFb in vitro. Finally, we established a K562 and KU-812 cells with diferent concentrations NOD/SCID mouse model bearing KU-812 tumors to fur- of wogonin were collected, the residual medium was ther verify the anticancer efect and potential mechanism washed with PBS, 0.5% BSA solution was added and of wogonin. blown evenly. Te non-specifc binding sites on the sur- face of the two cells were sealed, centrifuged and washed Materials and methods with PBS to remove the residual BSA solution. After dis- Compounds and regents persing the cell mass with 50 μL PBS, a certain amount of Wogonin (Purity > 99%) was isolated from Scutellaria antibody was added to fully bind the fuorescently labeled Baicalensis according to previously reported method antibody to the diferentiated antigen on the CML cell [28]. Wogonin was dissolved in dimethyl sulfoxide surface. Te antibody was incubated in a 37 °C shaker (DMSO, Sigma-Aldrich, Missouri, USA) at a concentra- in darkness for 50 min. Unbound antibody was washed tion of 0.1 M and stored at −80 °C. Ten the solution was with PBS and centrifuged. After resuspended with 400 μL diluted to the indicated concentration with RPMI-1640 PBS, the positive cell percentage was analyzed and calcu- or IMDM medium (GIBCO, CarlsBAD, CA, USA). FITC lated using Flowjo program. anti-human CD71 (Transferrin Receptor), PE anti-human Glycophorin A (CD235a), FITC anti-human CD41 and Western blot analysis PE anti-human CD61 antibodies were purchased from Total proteins were extracted from cells after wogonin eBioscience (San Diego, CA, USA). treatments for indicated time. Te cells were lysed in For in vivo experiments, wogonin was prepared as an RIPA bufer (Termo Scientifc, Massachusetts, USA) intraperitoneal injection administration formulation by with protease inhibitors (PMSF, NaF, leupeptin, and dith- Dr. Xue Ke at the College of Pharmacy, China Pharma- iothreitol) on ice for 50 min and then cell lysates were ceutical University. Imatinib Mesylate was purchased clarifed by centrifugation at 14,000 rpm (Eppendorf, from CHIA TAI TIANQING PHARMACEUTICAL Hamburg, Germany) for 20 min at 4 °C. Te concentra- GROUP CO.LTD (Nanjing, China) and prepared as an tion of proteins was quantifed with a BCA Protein Assay intragastric administration. Kit (Termo Fisher Scientifc, USA). Equal amounts of proteins were separated with 8–12% SDS-PAGE and Cell cultures transferred to the PVDF membranes (Millipore, Boston, Te chronic myeloid leukemia cell lines K562 and MA, USA). Membranes were blocked with 3% BSA and KU-812, human embryonic kidney cell line 293 T were incubated overnight at 4 °C with primary antibodies, fol- purchased from the Cell Bank of the Shanghai Institute lowed by incubation with HRP-coupled secondary anti- of Biochemistry & Cell Biology. Primary chronic mye- body for 1 h at room temperature. After washing the loid leukemia cells from newly diagnosed CML patients membranes with PBST three times, detection was per- (Te Afliated DrumTower Hospital, Nanjing, China) formed by AI600 imaging system (GE Healthcare, Pitts- were collected using lymphocyte-monocyte separation burgh, PA, USA). medium (Jingmei, Nanjing, China). K562 cells and pri- Primary antibodies specifc for RNA Pol II, p-RNA Pol mary CML cells were cultured in RPMI-1640 medium, II -Ser2, p-RNA Pol II -Ser5, MCL-1, Cyclin T1, FOG-1, KU-812 cells were cultured in IMDM medium, 293 T GAPDH and β-actin were obtained from ABclonal Tech- cells were cultured in DMEM medium, supplemented nology (Wuhan, China). Antibodies against CDK9, GATA-1 with 10% fetal bovine serum (FBS) (GIBCO, CarlsBAD, were obtained from Cell Signaling Technology (Danvers, USA), 100 U/ml of benzylpenicillin and 100 U/ml of MA). Antibody against RUNX1 was obtained from Protein- streptomycin in a humidifed environment with 5% ­CO2 tech (Wuhan, China). HRP Goat Anti-Rabbit IgG (H + L) at 37 °C. and HRP Goat Anti-Mouse IgG (H + L) secondary antibody were obtained from ABclonal Technology (Wuhan, China). Flow cytometric analysis of apoptosis Annexin V/PI staining assay was used to detect apoptosis Immunoprecipitation of cells after wogonin treatment. Te cells were collected Cell lysate was incubated with special antibody and 20 and stained with Annexin V/PI Cell Apoptosis Detection µL protein A/G-conjugated beads (MCE) at 4 °C over- Kit (Vazyme biotec, Nanjing, China) according to the night. After washing three times with PBS bufer, samples Qing et al. Cell Commun Signal (2021) 19:83 Page 4 of 16

were collected and re-suspended in 20 µL SDS-sample from the culture medium 48 h after transfection. And bufer (0.5 M Tris–HCl, pH 6.8, 20% glycerol, 2% SDS, then infected target cells with virus supernatant and 5% 2-mercaptoethanol and 4‰ bromophenol blue) and selected 2 μg/mL puromycin for screening. boiled for 10 min. Ten the samples were subjected to western blot with indicated antibodies. Quantitative real‑time RT‑qPCR RT-qPCR was performed according to the manufacturer’s Cell transfection and lentivirus package instructions [29]. Te primer sequences were as follows: For RNA interference by lentiviral vectors, CDK9 shRNA constructs and a negative control construct created in Human CDK9: forward: 5’- AAA​GTC​TGC​CAG​ the same vector system (pLKO.1) were purchased from CTT​CAG​GA -3’. Corues Biotechnology. Cells were co-transfected with reverse: 5’- ACT​TCT​GCG​AGC​ATG​ACC​TT -3’. shRNA constructs (10 μg) together with Lentiviral Mix Human GAPDH: forward: 5’-TGA​TGA​CAT​CAA​GAA​ (10 μL) and HG Transgene TM Reagent (60 μL), accord- GGT​GGT​GAA​ -3’. ing to the manufacturer’s instruction of Lentiviral Pack- reverse: 5’- TCC​TTG​GAG​GCC​ATG​TGG​GCCAT -3’. aging Kit (YEASEN). Viral supernatant was harvested

Fig. 1 Wogonin induced erythroid diferentiation in chronic myeloid leukemia cell lines and primary CML cells. a K562 and KU-812 cells were treated with wogonin (0, 5, 10, 20, 40 and 80 μM) for 120 h. The expression of CD41 and CD61 was detected by fow cytometry analyses. b Quantifcation of the expression of CD41/CD61. Columns represent the mean from three parallel experiments (mean SD), compared with control ± group. c K562 and KU-812 cells were treated with wogonin (0, 5, 10, 20, 40 and 80 μM) for 120 h. The expression of CD71 and GPA was detected by fow cytometry analyses. d Quantifcation of the expression of CD41/CD61. Columns represent the mean from three parallel experiments (mean SD). *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group. e #1 Primary CML cells were treated with wogonin (0, 20, 40 and ± 80 μM) for 120 h. The expression of CD71 and GPA was detected by fow cytometry analyses. f Quantifcation of the expression of CD41/CD61. Columns represent the mean from three parallel experiments (mean SD). *p < 0.05, **p < 0.01, compared with control group ± Qing et al. Cell Commun Signal (2021) 19:83 Page 5 of 16

Immunofuorescence analysis with standard laboratory food and water throughout the Immunofuorescence assays were performed as previ- experimental period. Te animal study was carried out ously reported [30]. Images were observed with a confo- according to the regulations of the China Food and Drug cal microscope (FV1000; Olympus, Tokyo, Japan). Administration (CFDA) on Animal Care.

TUNEL assay Apoptosis was determined using a One Step TUNEL FACS analysis of cells extracted from spleens apoptosis assay kit (Beyotime, Jiangsu, China). A double- Cells extracted from spleens were performed as previ- staining technique was used; TUNEL staining (green fuo- ously reported [32]. FACS Calibur fow cytometry was rescence) was used to quantitate apoptotic cell nuclei and used to detect huCD45 positive cells. In this model, DAPI staining (blue fuorescence) was used to quantitate human CD45 positive cells were defned as human leu- the total cell nuclei, as previously described [31]. Te kemia cells. stained samples were observed under a confocal micro- scope (FV1000; Olympus, Tokyo, Japan). Tree visual Statistical analysis felds were selected randomly for each specimen. All results are expressed as the mean ± SD from at least three independent experiments performed in a paral- lel manner. Statistical analyses of multiple group com- Animal models parisons were performed by one-way analysis of variance Te female NOD/SCID mice (5 weeks old, weighing followed by the Bonferroni post-hoc test. Comparisons 16–20 g) (Cavens Changzhou Laboratory Animal Co., Ltd, between two groups were analyzed using two-tailed Stu- Jiangsu, China) were sublethally irradiated (1.8 Gy) and were dent’s t tests. Te signifcance of diferences is indicated 6 as *p < 0.05, **p < 0.01 and ***p < 0.001. engrafted with KU-812 cells (6 × ­10 ) via subcutaneously injection within 24 h following the radiation treatment. After 10 days, the mice were divided randomly into three groups Result Wogonin induced erythroid diferentiation in chronic (n = 6 per group): a control group (0.9% normal saline), a wogonin-treated group (80 mg/kg) and an Imatinib-treated myeloid leukemia cell lines and primary CML cells group (200 mg/kg). Te animals in wogonin groups were In order to investigate anti-CML efects of wogonin, we intraperitoneal injection (i.p) of wogonin (80 mg/kg, every frst detect diferentiation on K562, KU-812 and primary day for 2 weeks). Te animals in Imatinib-treated group CML cells. K562 and KU-812 cells were treated with were received administrated orally of Imatinib (200 mg/kg, indicated concentrations of wogonin (0, 5, 10, 20, 40, every other day for 2 weeks). Te tumor volume of mice was 80 μM) for 120 h. We examined the expression of CD41 measured daily with an electronic vernier caliper. Finally, the and CD61, two megakaryocytic diferentiation specifc animals were sacrifced, and the tumors were prepared for markers. However, the numbers of CD41 and CD61 western blots and immunofuorescence assays, the spleens positive cells did not have signifcant increased after were prepared for fow cytometric analysis after the cells wogonin treatment in K562 and KU-812 cells (Fig. 1a, b). labeled with human CD45-PE (huCD45-PE) and the survival We then detected the expression of CD71 and GPA, two status are counted. Te staining of huCD45-PE is used to erythroid diferentiation specifc markers. As shown in determine the infltration of KU-812 cells. Fig. 1c, d, treatment with wogonin increased the propor- Animals were maintained in an air-conditioned and tion of CD71- and GPA-positive cells in K562 cells and KU-812 cells. In #1 primary CML cells, wogonin induced pathogen-free environment (23 ± 2 °C, 55 ± 5% humidity) under controlled lighting (12 h light/day) and supplied erythroid diferentiation in a concentration-dependent

(See fgure on next page.) Fig. 2 P-TEFb was involved in wogonin-induced erythroid diferentiation and regulated the protein interaction of GATA-1, RUNX1 and FOG-1. a After treatment with wogonin (0, 5, 10, 20, 40 and 80 μM) for 48 h in K562 and KU-812 cells, and in #1 primary CML cells wogonin (0, 20, 40, 80 μM) was treated for 48 h. Protein expression levels of p-RNA Pol II (Ser2), p-RNA Pol II (Ser5), RNA Pol II and CDK9 were analyzed by western blot. β-actin was used as a loading control. The results are representative of three independent experiments(mean SD). *p < 0.05, **p < 0.01, ***p < 0.001, ± compared with control group. b After treatment with wogonin (0, 5, 10, 20, 40 and 80 μM) for 48 h in K562 and KU-812 cells, and in #1 primary CML cells wogonin (0, 20, 40, 80 μM) was treated for 48 h. Protein expression levels of Cyclin T1 were analyzed by western blot. β-actin was used as a loading control. The results are representative of three independent experiments. (mean SD). *p < 0.05, **p < 0.01, compared with control group. ± c KU-812 cells were treated with wogonin (0, 5, 10 and 20 μM) for 72 h. Cells were immunoprecipitated with anti-GATA-1 antibody, followed by western blot analysis with anti-FOG-1 and anti-RUNX1 antibodies. GAPDH and β-actin were used as a loading control respectively. The results are representative of three independent experiments (mean SD). *p < 0.05, **p < 0.01, compared with control group ± Qing et al. Cell Commun Signal (2021) 19:83 Page 6 of 16

Fig. 2 (See legend on previous page.) Qing et al. Cell Commun Signal (2021) 19:83 Page 7 of 16

manner (Fig. 1e, f). We concluded that wogonin induced wogonin (0, 5, 10, 20, 40, 80 μM) for 48 h in KU-812 cells erythroid diferentiation but not megakaryocytic dif- (Fig. 2a). In K562 cells, the protein levels of RNA Pol II ferentiation in K562, KU-812 and primary CML cells as did not signifcantly change but its phosphorylated form judged by fow cytometry analyses. (Ser2 and Ser5) were signifcantly decreased after treat- ment with various concentrations of wogonin for 48 h P‑TEFb was involved in wogonin‑induced erythroid (Fig. 2a). Te expression of CDK9 slightly increased after diferentiation and regulated the protein interaction treatment of 80 μM wogonin on K562 but in KU-812 of GATA‑1, RUNX1 and FOG‑1 cells the expression of CDK9 had no statistically signif- P-TEFb is a general transcription factor that regulates cant diference (Fig. 2a). Similarly, in #1 primary CML transcription elongation through phosphorylation of cells wogonin reduced the expression of RNA Pol II and the C-terminal tail domain (CTD) of RNA polymerase its phosphorylation form (RNA Pol II Ser2 and Ser5), II (RNAP II), it is a heterodimer kinase composed of the level of CDK9 had slight increased by treatment CDK9 and its regulated subunit Cyclin T1 [33]. For veri- with 80 μM wogonin (Fig. 2a). Moreover, the expression fying whether erythroid diferentiation associated with of Cyclin T1 had no signifcant change in K562, KU-812 CDK9 on chronic myeloid leukemia cell lines and pri- and #1 primary CML cells (Fig. 2b). It is indicated that mary CML cells, we frst constructed lentiviral recom- wogonin inhibited the kinase activity of P-TEFb by binants expressing shRNA targeting CDK9 gene on K562 reducing the expression of the phosphorylation forms of cells and evaluated knock down efciency by using RT- RNA Pol II, which is a substrate protein of P-TEFb. qPCR (Additional fle 1: Fig. S1a). Ten we also tested the Some studies have shown that P-TEFb mediates the expression of RNA Pol II, the phosphorylation level of binding of GATA-1 and RUNX1, which promotes mega- RNA polymerase II (RNA Pol II Ser2 and Ser5) by west- karyocytic diferentiation and inhibits erythrocyte dif- ern blot. Results showed that the expression of phospho- ferentiation [35]. FOG-1 (Friend of GATA-1) specifcally rylation forms of RNA polymerase II Ser2 and Ser5 were binds to the N-terminal zinc fnger structure of GATA- both downregulated compared to negative control group, 1, the protein interaction of GATA-1 and FOG-1 plays it was indicated that P-TEFb kinase activity was inhib- an essential role in erythrocyte diferentiation [36]. ited when CDK9 was knocked down (Additional fle 1: Terefore, for further study the mechanism of wogonin- Fig. S1b). To further confrm the involvement of CDK9 induced erythroid diferentiation, co-IP was used to on erythroid diferentiation in CML cells, we detected investigate the protein interaction of GATA-1- FOG-1 the expression of surface antigens (CD71 and GPA) by and GATA-1-RUNX1 in KU-812 cells. As anticipated, fow cytometry analyses. Results showed that the ratio of wogonin increased the binding capacity of GATA-1 and erythroid diferentiation was increased to 56.85 ± 3.19% FOG-1, however wogonin dramatically decreased the in shCDK9 group compared to negative control group binding capacity of GATA-1 and RUNX1 (Fig. 2c). (Additional fle 1: Fig. S1c, d). In previous studies we have already known wogonin is Wogonin induced apoptosis in KU‑812 cells a CDK9 inhibitor, which directly binds to the ATP-bind- through downregulating MCL1 ing pocket of CDK9 [34]. In order to investigate P-TEFb To further investigate the efect of apoptosis in CML kinase activity involving in wogonin-induced erythroid cells, Annexin V/PI double-staining assay was conducted diferentiation, we examined the expression of the CTD in K562, KU-812 cells and #1 primary CML cells treated of RNA Pol II in these cells. Western blot showed that the by various concentrations of wogonin (0, 5, 10, 20, 40, protein levels of RNA Pol II had slightly decreased and its 80 μM) for 48 h. Results showed that high concentrations phosphorylated forms (Ser2 and Ser5) were signifcantly of wogonin (40, 80 μM) induced more obvious apoptosis decreased after treatment with various concentrations of in KU-812 cells, but did not in K562 and #1 primary CML

(See fgure on next page.) Fig. 3 Wogonin induced apoptosis in KU-812 cells through downregulating MCL1. a KU-812, K562 and #1 primary CML cells were treated with Wogonin (0, 5, 10, 20, 40 and 80 μM) for 48 h. Flow cytometric analysis of Annexin V/PI stained cells. Data represent the mean SD of three ± independent experiments. b Quantifcation of apoptosis cells. Columns represent the mean from three parallel experiments (mean SD). *p < 0.05, ± **p < 0.01, compared with control group. c The mRNA level of MCL1 in KU-812, K562 cells and #1 primary CML cells after cells were treated with wogonin (0 and 80 μM) for 48 h. Columns represent the mean from three parallel experiments (mean SD). **p < 0.01, compared with control ± group. d The protein expression level of MCL-1 in KU-812, K562 cells and #1 primary CML cells after cells were treated with various concentrations of wogonin for 48 h. β-actin and GAPDH was used as a loading control respectively. The results are representative of three independent experiments. e Quantifcation of the protein expression level of MCL-1 in KU-812, K562 cells and #1 primary CML cells. Columns represent the mean from three parallel experiments (mean SD). *p < 0.05, **p < 0.01, compared with control group ± Qing et al. Cell Commun Signal (2021) 19:83 Page 8 of 16

Fig. 3 (See legend on previous page.) Qing et al. Cell Commun Signal (2021) 19:83 Page 9 of 16

cells (Fig. 3a, b). Moreover, after treatment of wogonin and observed. Mice inoculated with KU-812 cells had sig- (80 μM) for 48 h, KU-812 cells were dyed for TUNEL- nifcantly enlarged spleens, and splenomegaly was efec- FITC, compared with control group, wogonin treatment tively alleviated in the treatment groups with wogonin and group was TUNEL-positive and the cells showed more Imatinib (Fig. 4e, f). We also collected spleens randomly green fuorescence, it was indicated that wogonin could for biopsy which were selected from each group. Results induce apoptosis in KU-812 cells (Additional fle 3: Fig. of immunofuorescence showed that compared with mice S3). Furthermore, as a downstream target gene of RNA in the blank group, the numbers of huCD45­ + cells in Pol II, MCL1 is closely related the survival and mainte- spleens of mice inoculated with KU-812 cells were signif- nance of tumor cells [33]. In order to examine the mRNA cantly increased in the control group, while the numbers level of MCL1, we frst used RT-PCR to detect the expres- of huCD45­ + cells in wogonin and Imatinib-treated group sion of MCL1 after treatment of wogonin. Results showed were signifcantly decreased (Fig. 4g). For investigating that 80 μM wogonin inhibited transcriptional activity of whether wogonin had obvious toxicity on mice, we there- MCL1 in KU-812 cells, but in K562 and primary CML fore assessed the efect of wogonin on animals’ weight and cells no signifcant change was shown (Fig. 3c). Moreover, main organs. As indicated in Additional fle 2: Fig. S2a, results of western blot showed that wogonin decreased there was a weight drop of mice in the Imatinib-treated the protein expression of MCL-1 in KU-812 cells in a group, while no signifcant weight loss was observed in concentration-dependent manner (Fig. 3d,e). Similar wogonin group. According to hematoxylin and eosin with the level of mRNA, wogonin had no efect on the staining of three organs from each mouse, we found that protein expression of MCL-1 in K562 and #1 prima ry wogonin exerted lower toxicity whereas Imatinib-treated CML cells (Fig. 3d, e). Terefore, the downregulation of group displayed injuries of varying intensity (Additional MCL1 would be the cause of wogonin-induced apoptosis fle 2: Fig. S2b). in KU-812 cells. Wogonin modulated P‑TEFb kinase activity in vivo Wogonin exhibited anti‑CML efects and exerted lower In order to verify the mechanism of wogonin-induced toxicity in vivo anti-CML activity in vivo, we detected P-TEFb kinase In order to confrm wogonin-induced anti-CML efects associated proteins in KU-812 transplanted tumors by in vivo, we constructed NOD/SCID mouse models western blot. Results showed that the expression of the engrafted with KU-812 cells via subcutaneous transplanta- phosphorylation forms of RNA polymerase II (p-RNA tion. Te animals in control and wogonin-treated groups Pol II Ser2 and Ser5) and MCL-1 were decreased in were intraperitoneal injection with solvent or wogonin wogonin-treated group compared to control group, (80 mg/kg, every day), and the animals in Imatinib-treated which was consistent with the results in vitro (Fig. 5a). group were administrated orally (200 mg/kg, every other Te expression of CDK9 and cyclin T1 did not have day). After administration for two weeks, we killed them signifcant changes in wogonin-treated group (Fig. 5a). and the graft tumors were taken out and weighed. As was Moreover, immunofuorescence experiments also con- indicated by tumor pattern, wogonin exhibited an observ- frmed the decreased efect of wogonin on the expression able tumor inhibition efect in KU-812 xenografts com- of p-RNA Pol II (Ser2), p-RNA Pol II (Ser5) and MCL-1 pared with the control group (Fig. 4a, b, c). Compared in KU-812 cells xenografts (Fig. 5b). with Imatinib-treated group, the efect of wogonin-treated For demonstrating erythroid diferentiation induced group was almost the same (Fig. 4a, b, c). Te expression by wogonin in vivo, we detected surface antigens CD71 of Ki67 also indicated that both of wogonin and Imatinib and GPA in cells extracted from tumor issues of KU-812 possessed strong anti-proliferation activity (Fig. 4d). More- cells-bearing NOD/SCID mice. Results showed that the over, the spleens of each group of mice were dissected GPA/CD71 positive ratio in wogonin-treated group

(See fgure on next page.) Fig. 4 Wogonin exhibits anti-tumor efects and exerted lower toxicity in vivo. In total, KU-812 cells (6 ­106 cells/mouse) were subcutaneously × inoculated into NOD/SCID mice. The mice were randomized into 6 groups (6 mice per group), and treated with wogonin (80 mg/kg), Imatinib (200 mg/kg) and 0.9% normal saline for 2 weeks. a The tumor volume was measured and calculated every other day. Data represent mean SD ± of three independent experiments. *p < 0.05, **p < 0.01, compared with control group. b The resulting tumors excised from the animals after treatment. c The tumor weights in three groups were compared. Quantifcation of the data shown. Data represent mean SD of three independent ± experiments. *p < 0.05, **p < 0.01, compared with control group. d Efect of wogonin on expression of Ki67 in KU-812 xenografts (Original magnifcation 600). e–f Wogonin inhibits the splenomegaly of KU-812-bearing NOD/SCID mice. The typical photos of the spleen. Efect of × wogonin on the weight of spleen in KU-812 cells-bearing NOD/SCID mice. Each data point represents the mean SD of four animals for each group. ± **p < 0.01, ***p < 0.001, compared with control group. g Efect of wogonin on expression of ­huCD45+ in spleen of KU-812-bearing NOD/SCID mice. Spleen samples from three mice of each group (Original magnifcation 600) × Qing et al. Cell Commun Signal (2021) 19:83 Page 10 of 16

Fig. 4 (See legend on previous page.)

that wogonin could reduce the growth of CML cells by increased from 9.4 ± 0.36% to 34.60 ± 1.57% compared with control group, but in Imatinib-treated group no modulating P-TEFb kinase in vivo. efect was observed (Fig. 5c, d). All these results indicated Qing et al. Cell Commun Signal (2021) 19:83 Page 11 of 16

Discussion Ser5) of RNA pol II were also decreased in CDK9-shRNA Although classic TKIs targeting BCR-ABL1 in CML cells group. It was indicated that P-TEFb kinase activity was makes patient’s condition acquired greatly improved, but associated with erythroid diferentiation in CML cells. is not curative and frequently limited by intolerance or Ten we found wogonin decreased the phosphorylated resistance [37]. Diferentiation therapy remains a classi- forms (Ser2 and Ser5) of RNA pol II in a concentration- cal and unsurpassable therapeutic schedule in hemato- dependent manner but did not change the expression of logic malignancies [38]. All-trans retinoic acid in treating cyclin T1 in CML cell lines and primary CML cells. It was acute promyelocytic leukemia (APL) results in cure rates indicated that wogonin inhibited P-TEFb activity in CML of over 80% [39]. In this study, we found wogonin, a tra- cells. Moreover, co-immunoprecipitation assay showed ditional favonoid, induced diferentiation and apopto- that wogonin decreased the interaction of GATA-1 and sis in CML cells lines and primary CML cells mediated RUNX1 but increased the interaction of GATA-1 and by P-TEFb. Moreover, wogonin inhibited the growth of FOG-1. Te results demonstrated that wogonin pro- KU-812 xenografts mediated by P-TEFb was also verifed moted erythroid diferentiation but repressed mega- in vivo. karyocyte diferentiation, which could attribute to the P-TEFb complex is a special kinase that plays a key inhibition of P-TEFb activity. We also verifed the inhibi- role in transcription elongation. RNA pol II is the main tion of P-TEFb activity in vivo, meanwhile, erythroid dif- functional target of the complex. GATA-1, which is a key ferentiation was also increased by wogonin in xenograft transcription factor that interacts with a variety of pro- of KU-812 cells. teins including FOG-1 and RUNX1 to regulate erythroid Te transcription of protein with short half-lives like and megakaryocyte diferentiation respectively [35, 40]. MCL-1 is prolonged via P-TEFb, which phosphoryl- However, FOG-1 negatively regulates RUNX1-GATA-1 ates the carboxy terminal of RNA polymerase II [33, 42]. cooperation, which attains the switch between erythroid CDK9 is a critical component of P-TEFb. In this context, and megakaryocyte diferentiation [35]. Te interac- inhibition of CDK9 by wogonin blocked the activity of tion of GATA-1 with RUNX1 is highly dependent on the RNA pol II and induced apoptosis in KU-812 cells. As P-TEFb kinase complex [22, 35]. It has been reported that the downstream of RNA pol II, the transcriptional level wogonin induced erythroid diferentiation in CML cells of MCL-1 was decreased in KU-812 cells. Consistent with [41]. CDK9 is a core component of P-TEFb by binding that the protein level of MCL-1 also decreased in KU-812 with cyclinT1 to form an active P-TEFb kinase complex. cells. Moreover, we verifed the decreased expression of Previous studies have shown that wogonin has a good MCL-1 in in KU-812 cells xenografts. It’s worth mention inhibitory efect on CDK9 kinase [34]. To this end, we that the protein expression of MCL-1 was not signifcant suspected that wogonin induced erythroid diferentiation decreased in vivo of Imatinib treated group (Fig. 5a, b). in CML cells involving modulation on P-TEFb. In general, some studies showed MCL-1 is a BCR-ABL GATA-1 is the key node of megakaryocyte / erythroid dependent target in CML cells and TKIs could decreased diferentiation, and the formation of GATA-1/RUNX1 the expression of MCL-1 [43]. However, heterogeneity of complex, which is necessary for megakaryocyte difer- tumors in vivo study may cause abnormal expression of entiation, depends on the activity of P-TEFb. However, MCL-1 in Imatinib treated group. As a matter of fact, we whether inhibition of P-TEFb activity can promote eryth- found some studies showed MCL-1 expression elevated roid diferentiation remains unknown. Tus, we frst used after Imatinib compared with control group in CML CDK9-shRNA to knock down the expression of CDK9 patients, which was linked to the drug resistance and and results showed that erythroid diferentiation was individual diference [44]. increased in CDK9-shRNA group compared to scram- In K562 and #1 primary CML cells, wogonin could not ble group. Moreover, the expression of the downstream induce apoptosis and decrease the expression of MCL- target of CDK9, the phosphorylated forms (Ser2 and 1. We observed that in K562 cells wogonin-induced

(See fgure on next page.) Fig. 5 Wogonin modulated P-TEFb kinase activity in vivo. a The protein expression of p-RNA Pol II (Ser2), p-RNA Pol II (Ser5), CyclinT1, CDK9 and MCL-1 in KU-812 xenografts. β-actin was used as a loading control. The results are representative of three independent experiments (mean SD). ± *p < 0.05, **p < 0.01, compared with control group. b Immunofuorescence and stained with p-RNA Pol II (Ser2) (green fuorescence), p-RNA Pol II (Ser5) (green fuorescence) and MCL-1 (green fuorescence) respectively, as well as DAPI (blue fuorescence) in tumor of KU-812-bearing NOD/ SCID mice. They were detected by confocal microscopy (FV1000; Olympus) with FV10-ASW2.1 acquisition software (Olympus) at room temperature (Original magnifcation 600). c The expression of CD71 and GPA was detected in tumor of KU-812-bearing NOD/SCID mice by fow cytometry × analyses. d Quantifcation of the expression of CD71/GPA. Data represent mean SD of three independent experiments. ***p < 0.001, compared ± with control group Qing et al. Cell Commun Signal (2021) 19:83 Page 12 of 16

Fig. 5 (See legend on previous page.) Qing et al. Cell Commun Signal (2021) 19:83 Page 13 of 16

Fig. 6 Graphical abstract on the mechanisms of pharmacologic targeting of the P-TEFb complex induced by wogonin on CML cells

erythroid diferentiation was concentration-dependent can be fully distinguished notably regarding their sensi- but in KU-812 cells, the degree of erythroid diferentiation tivity to caspase and p38 MAP kinase inhibition. [47, 48]. was gradually increased by the concentration of 0, 5, 10, It has been reported that overexpression of MCL1 may 20 μM but decreased when the concentration was 40 and inhibit apoptosis and subsequent inhibition of apoptosis 80 μM. Although wogonin inhibited the kinase activity of may support cellular cell diferentiation of CML cells [49]. P-TEFb in CML cell lines and primary CML cells, MCL-1 An investigation showed that one murine cell line, ELM- is one of downstream target genes of RNA Pol II. As a I-1, required both activin A and EPO to achieve erythroid matter of fact, a number of growth factors, cytokines and diferentiation, however, apoptosis was induced by treat- cytotoxic stimuli (e.g. drugs, radiation) regulate MCL-1 ment with activin A alone. On the contrary, in another transcription through cell-type dependent efects on sig- murine cell line, F5-5, diferentiation was sufciently nal transduction pathways such as the PI3K/Akt, JAK/ induced by activin A alone to achieve an erythroid lineage. STAT, p38/MAPK, and MEK/ERK pathways, with both One possible explanation for these diferences in activin A antiapoptotic and proapoptotic stimuli involved [45, 46]. reactivity in these two cell lines was the state of the EPO Tus not only RNA Pol II but also diferent types of signal- receptor [50]. As observed in the case of wogonin in three ing pathways may be involved in the regulation of MCL-1 types of cells, the diferences among these cells like the expression, which depends on the cell types. In our study, state of the EPO receptor or cell sensitivity to wogonin we speculated that wogonin-induced diferent efects on may cause the diferent response degree in diferent cells, MCL-1 may be regulated by diferent signaling pathways, resulting in the transition of cell death and diferentiation. which is not limited to RNA Pol II. Some studies showed Te specifc mechanism of the transformation of these that inhibiting apoptosis by ZVAD-fmk allows the surviv- two efects remains to be further explored. ing cells to diferentiate towards the erythroid lineage, to Taken together, our results showed the involvement of some extent, BCR-ABL1-mediated apoptosis and eryth- P-TEFb in wogonin-induced apoptosis and erythroid dif- roid diferentiation are two independent processes which ferentiation in CML cells. Furthermore, we demonstrated Qing et al. Cell Commun Signal (2021) 19:83 Page 14 of 16

that wogonin induced-erythroid diferentiation was asso- Authors’ contributions YJQ and XYW designed and performed research, analyzed data, and wrote the ciated with the increasing interaction of GATA-1 and paper; HZW and PH performed research and analyzed data; HL and XXY per- FOG-1, which were regulated by P-TEFb. Tis research formed research; MYZ and ZYW collected data and performed statistical analy- indicated that wogonin showed good inhibitory efect on sis; JYX provided the blood samples; YZ, JYX performed statistical analysis; and QLG and HH conceptualized the project and directed the experimental design CML cells through this unique mechanism, suggesting data analysis. All authors read and approved the fnal manuscript. the potential application of wogonin in developing into a novel agent for the treatment of CML (Fig. 6). Funding The work was supported by the Nation Natural Science Foundation of China (81873046, 81830105, 81903647, 81503096, 81673461), the Drug Innovation Major Project (2017ZX09301014, 2018ZX09711001-003-007, 2017ZX09101003- Conclusions 005-023), Natural Science Foundation of Jiangsu province (BK20190560, BE2018711), Nanjing Medical Science and Technology Development Project Our research elucidated the regulatory mechanism of (YKK17074, YKK19064), Research and Innovation Project for College Graduates P-TEFb and GATA-1 signal crosslinking in the erythroid of Jiangsu Province (KYCX18_0803), China Postdoctoral Science Foundation diferentiation of CML cells induced by wogonin. Moreo (No. 2018M642373) and “Double First-Class” University project (CPU2018GF11, - CPU2018GF05). ver, we demonstrated that wogonin induced erythroid diferentiation of CML cells by inhibiting P-TEFb kinase Availability of data and materials activity, reducing the interaction between GATA-1 and The datasets supporting the conclusions of this article are included within the article and its additional fles. RUNX-1, and promoting the binding of GATA-1 to FOG- 1. Tis study highlighted a role of P-TEFb in wogonin- Declarations treated CML cells and provided a potential mechanism for the application of wogonin. Ethical approval and consent to participate The animal study was carried out according to the regulations of the CFDA on Animal Care. All patients’ samples were collected after informed consent in Abbreviations accordance with the Declaration of Helsinki. The study was approved by the CML: Chronic myeloid leukemia; P-TEFb: The positive transcription elonga- Ethics Committee of China Pharmaceutical University. tion factor b; CDK9: Cyclin-dependent kinase 9; CDKs: Cyclin-dependent kinases; RNA Pol II: RNA Polymerase II; CTD: C-Terminal Domain; APL: Acute Consent for publication promyelocytic leukemia; RA: Retinoic acid; CMP: Common myeloid progenitor; Not applicable. MEP: Megakaryocyte erythrocyte progenitor; GRA​: Granulocyte macrophage progenitor; GATA-1: GATA binding protein 1; FOG-1: Friend of GATA-1; Competing interests RUNX1: Runt-related transcription factor 1; RT-qPCR: Real-time quantitative Authors declare no conficts of interest for this article. polymerase chain reaction; TUNEL: TdT-mediated dUTP nick-end labeling; HSC: Hematopoietic stem cell. Author details 1 State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcino- genesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Supplementary Information Nanjing 210009, People’s Republic of China. 2 Department of Pharmacology, The online version contains supplementary material available at https://​doi.​ School of Medicine and Holostic Integrative Medicine, Nanjing University 3 org/​10.​1186/​s12964-​021-​00764-5. of Chinese Medicine, Nanjing 210023, People’s Republic of China. Depart- ment of Hematology, The First Afliated Hospital of Nanjing Medical Univer- sity, Jiangsu Province Hospital, Nanjing 210029, People’s Republic of China. Additional fle 1. Figure S1. CDK9 shRNA K562 cells enhanced the rates 4 Department of Hematology, The Afliated DrumTower Hospital of Nanjing of erythroid diferentiation. (a) K562 cells were transfected with NC shRNA University Medical School, Nanjing 210008, People’s Republic of China. or CDK9 shRNA. The mRNA level of CDK9 was detected by RT-PCR. Data represent mean SD of three independent experiments. ***p < 0.001, Received: 3 March 2021 Accepted: 2 July 2021 compared with control± group. (b) In control, NC and shCDK9 groups, cell lysates were analysed for p-RNA Pol II (Ser2), p-RNA Pol II (Ser5), RNA Pol II and CDK9 expression by western Blot. Data represent mean SD of three independent experiments. *p < 0.05, **p < 0.01, compared with± NC group. (c) In control, NC and shCDK9 groups, the expression of CD71 and GPA References was detected by fow cytometry analyses. Data represent the mean SD 1. Horne GA, Stobo J, Kelly C, Mukhopadhyay A, Latif AL, Dixon-Hughes J, ± of three independent experiments. (d) Quantifcation of the expression of McMahon L, Cony-Makhoul P, Byrne J, Smith G, et al. A randomised phase CD71/GPA. Data represent mean SD of three independent experiments. II trial of hydroxychloroquine and imatinib versus imatinib alone for ± ***p < 0.001, compared with NC group. patients with chronic myeloid leukaemia in major cytogenetic response Additional fle 2. Figure S2. Toxicological assessment. (a) NOD/SCID mice with residual disease. Leukemia. 2020;34:1775–86. weights were recorded every 2 days. (b) H&E stained main organs of mice 2. Abduelkarem AR, Anbar HS, Zaraei SO, Alfar AA, Al-Zoubi OS, Abdelkarem from treated and control group to evaluate the toxicity of wogonin. EG, El-Gamal MI. Diarylamides in anticancer drug discovery: A review of pre-clinical and clinical investigations. Eur J Med Chem. 2020 Feb Additional fle 3. Figure S3. Apoptosis detection of KU-812 cells by 15;188:112029. TUNEL (a) KU-812 cells were treated with wogonin (0, 80 μM) for 48 h. Cell 3. Kantarjian HM, O’Brien S, Anderlini P, Talpaz M. Treatment of myelog- apoptosis was measured by TUNEL staining used a confocal microscope. enous leukemia: current status and investigational options. Blood. Three visual felds were selected randomly for each specimen. 1996;87:3069–81. 4. Karvela M, Baquero P, Kuntz EM, Mukhopadhyay A, Mitchell R, Allan EK, Chan E, Kranc KR, Calabretta B, Salomoni P, et al. ATG7 regulates energy Acknowledgements metabolism, diferentiation and survival of Philadelphia-chromosome- Thanks for all participants involved in this research. positive cells. Autophagy. 2016;12:936–48. Qing et al. Cell Commun Signal (2021) 19:83 Page 15 of 16

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